Evaluation of Viral Interference with MHC Class I-Restricted Antigen Processing and Presentation Using a Flow Cytometry-Based Approach

Abstract

The peptide content of MHC class I molecules present at the cell surface is monitored by surveilling CD8⁺ cytotoxic T cells. In case of a viral infection, a proportion of the MHC class I molecules will carry peptides derived from viral proteins. This allows the CD8⁺ T cells to recognize and eliminate virus-infected cells. This highly sensitive detection system of the host is counteracted by viruses, which have acquired functions to downregulate cell surface expression of MHC class I molecules. In this chapter, we describe a flow cytometry-based method to identify viral gene product(s) responsible for evasion from MHC class I-restricted antigen presentation. To this end, cells are transiently transfected using polyethylenimine (PEI) as a transfection reagent, followed by cell surface staining with MHC class I-specific monoclonal antibodies. Once viral proteins responsible for MHC class I downregulation have been identified, their mechanism of action can be characterized. Identification and characterization of virus-encoded MHC class I inhibitors augments our understanding of virus–host interactions and often provides new insights into antigen processing and presentation pathways, including related cellular processes such as protein trafficking and degradation.